Most human tumors are treated locally with ionizing radiation. Despite advances in the complex development and use of high energy linear accelerators, intricate computer-assisted treatment planning systems, which maximize the dose of radiation administered to a tumor, and the use of different and, perhaps, more efficient types of radiation, such as high linear energy transfer (LET) radiation, local control for a vast number of solid tumors has not substantially improved.
Over the past 50 years, radiobiologists have sought to gain a better understanding of the biological and physiological factors of tumors that might be important and exploitable in improving radiation-based cancer treatment. The biological characteristics of human tumors, however, are complex and poorly understood.
Oxygen has long been known to enhance the effectiveness of radiation. The presence of oxygen was shown to enhance the killing of cells in culture with X-rays by a factor of 2-3 (Howard-Flanders, Nature, 180, 1191-1192 (1957)). In other words, if cells are irradiated in an aerobic (.about.20% oxygen) as opposed to hypoxic (&lt;0.5% oxygen) environment, they are 2-3 times more susceptible to the cytotoxic effects of radiation. The term Oxygen Enhancement Ratio (OER) is used to describe the decreased radiosensitivity of cells under hypoxic conditions as opposed to aerobic conditions. The OER for mammalian cells exposed to X-rays under aerobic conditions is about 1, while the OER for mammalian cells exposed to X-rays under hypoxic conditions is about 2-3, representing the fact that hypoxic cells are about 2-3 times more resistant, i.e., less susceptible, to the cytotoxic effects of radiation as compared to aerobic cells.
It has been postulated that radiation produces carbon-centered radicals, probably in the cellular DNA, that can react with molecular oxygen, which is a diradical molecule, to yield a lesion that is toxic if not repaired, will result in cell death (yon Sonntag, The Chemical Basis of Radiation Biology, Taylor and Francis, eds., London, 1987). Under hypoxic conditions, very few, if any, oxygen-related lesions are formed, and, hence, there is less cell killing.
In 1956, Thomlinson and Gray (Br. J. Cancer, 9, 539-549 (1955)) observed in biopsied tumors taken from lung cancer patients that distinct regions of viable tumors were always near or surrounding blood capillaries. Regions of necrosis were found with increasing distances from the capillaries. Based on these observations, Thomlinson and Gray calculated that the maximum distance that oxygen could diffuse through actively metabolizing tissue was between 150 and 200 .mu.m. This distance coincided with actual measurements of biopsied tissue. This led Thomlinson and Gray to hypothesize that a gradient of oxygen concentration may exist as a function of distance from capillaries and that tumor cells located at 150-200 .mu.m away, while still viable, are in an environment of extremely low oxygen concentration. Such cells were considered to be resistant to radiation and their presence was thought to limit the effectiveness of radiation cancer treatment. Powers and Tolmach (Nature, 197, 710-711 (1963)) quantitatively showed, using a rat tumor model, that viable radiation-resistant hypoxic cells do exist in vivo.
The success of the local treatment of many cancerous tumors with ionizing radiation is believed to be limited by the presence of hypoxic cell subpopulations within the tumor (Thomlinson and Gray, supra; Powers and Tolmach, supra; and Gatenby et al., Int. J. Radiat. Oncol. Biol. Phys., 14, 831-838 (1988)). Given that hypoxic cells are approximately three-fold more resistant to radiation than aerobic cells, a major research objective of radiation oncology and biology has been to identify approaches to sensitize and eliminate hypoxic cells from tumors (Hall, "The oxygen effect and reoxygenation," in Radiobiology for the Radiologist, J. B. Lippincott Co., Philadelphia, Pa., pp. 137-160 (1988)). The goal has been to identify an approach that will reduce the OER from about 3.0 to about 1.0, i.e., so that hypoxic cells will have the same response to radiation as aerobic cells.
Over the course of 25 years, various in vitro systems have been used to evaluate agents for their potential to sensitize hypoxic cells to radiation (Hall et al., Br. J. Radiol., 39, 302-307 (1966); Bedford et al., Br. J. Radiol., 39, 896-900 (1966)). Such studies have demonstrated that nitric oxide gas sensitizes hypoxic bacterial cells to ionizing radiation (Howard-Flanders, supra). Nitric oxide (NO), which is nontoxic in the absence of radiation, was shown to have an affinity similar to that of oxygen and to "fix" radiation-induced carbon-centered radicals in DNA.
NO also has been shown to have vasodilatory effects on vasculature (Ignarro, Annu. Rev. Pharmacol. Toxicol., 30, 535-560 (1990)). Tumor blood flow was selectively reduced in tumor versus normal tissue by the administration of nitric oxide synthase, an enzyme that generates NO Andrade et al., Brit. J. Pharm., 104, 1092-1095 (1992)). In vivo studies have demonstrated, though, that the administration of high concentrations of NO gas to hypoxic cell targets has serious drawbacks, including damage to the lungs and the destruction of NO prior to arrival at the target cells by other chemical reactions, such as the diffusion-controlled oxidation of oxyhemoglobin in the blood. Accordingly, although NO gas may be of limited utility in the local treatment of lung tumors, its usefulness in the treatment of distant solid tumor sites is limited by an inability to deliver adequate concentrations of NO to the target site.
High LET radiation, i.e., neutron irradiation, has been proposed as an alternative to X-rays because hypoxic cells are less resistant to neutron radiation (OER=1.7) than x-rays (Hall, in Radiobiology for the Radiologist, J. B. Lippincott Co., Philadelphia, Pa., pages 161-177 (1988)). The use of neutrons in clinical radiotherapy, however, has not yielded significant improvement in tumor response and, due to the cost of constructing and maintaining a clinical neutron facility, probably will not be given further consideration.
The use of hyperbaric oxygen in the treatment of tumors also has been evaluated, both experimentally and clinically. Basically, hyperbaric oxygen (100%) is forced into hypoxic regions of tumors. This approach has shown some degree of hypoxic radiosensitization in rodent tumors (Powers and Tolmach, supra). However, multiple hyperbaric oxygen treatments, i.e., 20-30, are required, and very few patients have accrued for clinical trials.
Another approach that has received attention is the development of chemicals, in particular nitroimidazoles, which are not metabolized, like oxygen, but diffuse into hypoxic regions in tumors and sensitize the hypoxic cells to radiation (Adams et al., Radiat. Res., 67, 9-20 (1976)). At concentrations of about 1 mM, nitroimidazoles have been shown to radiosensitize (OER=1.6) hypoxic cells but not aerobic cells. The mechanism of hypoxic cell radiosensitization by nitroimidazoles is unknown. However, it is hypothesized that nitroimidazoles have similar electron affinity to that of oxygen and that, under hypoxic conditions, they can react with carbon-centered free radicals produced by radiation and, like oxygen, "fix" the damage on molecules that are necessary for cell survival. Two nitroimidazoles, namely misonidazole and SR-2508, have been introduced into clinical radiotherapy trials. Misonidazole was shown to be toxic to normal tissues, causing peripheral neuropathy, which limits the effective dosage (Phillips et al., Cancer Treat. Rep., 68, 291-301 (1984)). SR-2508 was specifically synthesized to circumvent this problem, and patients are currently being accrued to evaluate it as a hypoxic cell radiation sensitizer (Coleman et al., Int. J. Radiat. Oncol. Biol. Phys., 12, 1105-1108 (1986)).
An NO releasing agent, namely SIN-1, has been described as an agent that enhances the radiation sensitivity of a transplantable murine tumor (Wood et al., Biochem. Biophys. Res. Commun., 192, 505-510 (1993)). However, its usefulness as a hypoxic radiosensitizer is limited by its dependence on the presence of oxygen (Feelisch et al., J. Cardiovasc. Pharmacol., 14 (Suppl. 11), S13-S22 (1989); Bohn et al., J. Cardiovasc. Pharmacol. 14 (Suppl. 11), S6-S12 (1989)).
Adams et al. (supra) found that a correlation existed between the radiosensitizing activity of various nitroaromatic and nitroheterocyclic compounds and their electron affinity. Nitrobenzoyl derivatives of spermine and spermidine were shown to have increased hypoxic cell radiosensitization over misonidazole. The radiosensitizing activity was found to be attributable to the nitrobenzoyl group only, given that spermine and spermidine without the nitrobenzoyl group failed to demonstrate any sensitizing properties whatsoever (Murayama et al., Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med., 44, 497-503 (1983)). The usefulness of nitrobenzoyl derivatives of spermine and spermidine, however, is limited to a certain degree by relatively short half-lives in solution.
In view of the inadequacy of the various treatment modalities currently available to overcome the decreased sensitivity of hypoxic tumor cells to radiation, there remains a need for a method of sensitizing hypoxic cells to radiation. It is an object of the present invention to provide such a method. It is a related object of the present invention to provide a method of delivering nitric oxide to hypoxic cells in a tumor. It is another object of the present invention to provide a method of delivering nitric oxide to hypoxic cells by means of an agent, in particular a water-soluble agent, that spontaneously releases NO under physiological conditions without requiring the presence of oxygen. It is a further object of the present invention to provide for such delivery in a controlled and predictable manner.
Another object of the present invention is to provide a method of protecting noncancerous cells or tissues from radiation. It is a related object of the present invention to provide a method of delivering nitric oxide to noncancerous cells or tissues. It is another object of the present invention to provide a method of delivering nitric oxide to noncancerous cells or tissues by means of an agent, in particular a water-soluble agent, that spontaneously releases NO under physiological conditions without requiring the presence of oxygen. It is a further object of the present invention to provide for such delivery in a controlled and predictable manner.
Yet another object of the present invention is to provide a method of sensitizing cancerous cells to chemotherapeutic agents. It is a related object of the present invention to provide a method of delivering nitric oxide to cancerous cells. It is another object of the present invention to provide a method of delivering nitric oxide to cancerous cells by means of an agent, in particular a water-soluble agent, that spontaneously releases NO under physiological conditions without requiring the presence of oxygen. It is a further object of the present invention to provide for such delivery in a controlled and predictable manner. A further object of the present invention is to provide a method of protecting noncancerous cells or tissues from chemotherapeutic agents. It is a related object of the present invention to provide a method of delivering nitric oxide to noncancerous cells or tissues. It is another object of the present invention to provide a method of delivering nitric oxide to noncancerous cells or tissues by means of an agent, in particular a water-soluble agent, that spontaneously releases NO under physiological conditions without requiring the presence of oxygen. It is a further object of the present invention to provide for such delivery in a controlled and predictable manner. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.